RESUMO
Extracellular ATP released from pancreatic ß-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic ß-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic ß-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.
Assuntos
Glucose/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Pirofosfatases/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Glucose/farmacologia , Humanos , Secreção de Insulina , Células Secretoras de Insulina/química , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pirofosfatases/análise , Pirofosfatases/antagonistas & inibidores , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet ß-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing. A clinical study with 3 administered to subjects with T2DM provided proof of concept of 3 as a potential glucose-lowering therapy. This manuscript summarizes the scientific rationale, medicinal chemistry, preclinical, and early development data of this new class of GPR40 agonists.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Hipoglicemiantes/farmacologia , Piperidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Compostos de Espiro/farmacologia , Animais , Relação Dose-Resposta a Droga , Teste de Tolerância a Glucose , Células HEK293 , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Ratos , Ratos Zucker , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Oxytocin (OT) has been implicated in the cardiovascular responses to exercise, stress, and baroreflex adjustments. Studies were conducted to determine the effect of genetic manipulation of the OT gene on blood pressure (BP), heart rate (HR), and autonomic/baroreflex function. OT knockout (OTKO -/-) and control +/+ mice were prepared with chronic arterial catheters. OTKO -/- mice exhibited a mild hypotension (102 +/- 3 vs. 110 +/- 3 mmHg). Sympathetic and vagal tone were tested using beta(1)-adrenergic and cholinergic blockade (atenolol and atropine). Magnitude of sympathetic and vagal tone to the heart and periphery was not significantly different between groups. However, there was an upward shift of sympathetic tone to higher HR values in OTKO -/- mice. This displacement combined with unchanged basal HR led to larger responses to cholinergic blockade (+77 +/- 25 vs. +5 +/- 15 beats/min, OTKO -/- vs. control +/+ group). There was also an increase in baroreflex gain (-13.1 +/- 2.5 vs. -4.1 +/- 1.2 beats x min(-1) x mmHg(-1), OTKO -/- vs. control +/+ group) over a smaller BP range. Results show that OTKO -/- mice are characterized by 1) hypotension, suggesting that OT is involved in tonic BP maintenance; 2) enhanced baroreflex gain over a small BP range, suggesting that OT extends the functional range of arterial baroreceptor reflex; and 3) shift in autonomic balance, indicating that OT reduces the sympathetic reserve.